The goal of this project is to optimize genetic stability, and to control the survival, of genetically modified microorganisms for bioremediation applications. A major limitation with the use of genetically modified microorganisms for bioremediation is the inability to adequately control mutation and survival rates of the organisms during remediation. This leads to concerns with the environmental release of genetic materials. To address this barrier, tightly controlled systems are needed. The bacterium, Pseudomonas putida, will be genetically modified with a streptavidin- based suicide system to control survival, coupled to a catabolic plasmid for aromatic hydrocarbon degradation. Our preliminary data demonstrate that this system can achieve the required goals of genetic stability and control of survival in controlled laboratory experiments. Phase I of this project will focus on relocating the plasmid-borne control system onto the chromosome to further enhance stability of the genetically engineered organism. Phase II will address additional enhancements to this system, including the incorporation of a dual suicide system for further control of stability and survival, the addition of metal sequestration capabilities in the organism, and methods to rapidly monitor the organism in the environment through the streptavidin tracer. PROPOSED COMMERCIAL APPLICATIONS: Genetically engineered microorganisms have strong potential to address hazardous waste contamination problems in the environment. However, a major need is to control the survival of these organisms to mitigate concerns with their uncontrolled release. The project will address the need for tight control of these organisms through the use of a regulatory system to control cell survival tied to catabolic activity and facile monitoring capability.